Transcriber’s notes:

Apart from the following corrected misspellings the text of this book has been preserved as in the original:
xolol → xylol
side → slide
overstraining → overstaining

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SECTION CUTTING AND STAINING A PRACTICAL INTRODUCTION TO HISTOLOGICAL METHODS FOR
STUDENTS AND PRACTITIONERS

BY

W. S. COLMAN, M.D., M.R.C.P.

ASSISTANT PHYSICIAN (FORMERLY PATHOLOGIST) TO THE NATIONAL
HOSPITAL FOR THE PARALYSED AND EPILEPTIC; AND TO
THE HOSPITAL FOR SICK CHILDREN, GREAT
ORMOND STREET, ETC.

SECOND EDITION

ENLARGED AND IN MOST PART RE-WRITTEN

LONDON
H. K. LEWIS, 136 GOWER STREET, W.C.
1896

PRINTED BY
H. K. LEWIS, 136 GOWER STREET,
LONDON, W.C.

PREFACE TO THE SECOND EDITION.

In preparing this edition I have endeavoured to meet the requirements of students, and of practitioners who desire to keep up their histological work. Those methods are selected which have been found to work well in practice, and it has been thought better to describe a few in detail rather than give a short account of many similar methods.

I have again to express my obligation to the various instrument makers for the illustrations of microtomes, &c.; to Dr. Fearnley, of Bradford, for the description of his method for injecting blood vessels, and to Messrs. Macmillan and Co. for permission to copy figures 10 and 11.

W. S. COLMAN.

Wimpole Street, W.
Sept., 1896.

CONTENTS.

SECTION CUTTING AND STAINING.

CHAPTER I.

Apparatus Required.

Probably there is nothing more perplexing to a beginner than to decide what apparatus is required. If he consult a price list, it is difficult for him to tell which articles will be necessary, and which will be either luxuries, or required only for special investigation.

In the following account of requisites, those only will be described which it is useful to have always at hand. They will be found sufficient for ordinary work, but for special investigations a more elaborate equipment will be required.

All staining and other reagents should be made as far as possible by the worker himself, according to the directions given in later chapters. This should at any rate be done at first, as the knowledge thus gained will prove invaluable. It will also effect a great saving if articles that are used in any quantity, such as methylated spirit, distilled water, &c., are bought by the gallon, and not in small quantities.

Almost all the processes described here can be carried out without the use of a fully equipped laboratory, in fact, in an ordinary room. The only furniture required is a firm table, and a cupboard and shelves for storing reagents.

The following should also be procured:—

Jars or bottles, with well fitting stoppers or corks, to contain the tissues while being hardened. They should not hold less than two ounces. Empty drug bottles which can usually be obtained from druggists for a few pence, serve very well.

Smaller bottles should also be procured for keeping specimens in spirit after they have been hardened until one is ready to cut sections. After sections have been cut from a portion of the specimen, the rest should be preserved, in case it is wanted for further investigation. Each specimen must be labelled, with a name or a number corresponding to a reference in the note-book, and a large number of specimens may then be kept in the same jar. The best way to label them is to write the name or number on a piece of vegetable parchment in ordinary “marking ink,” and warm it until the writing is black. The little label should then be fixed to a corner of the piece of tissue with a stitch or by a fine pin, and it may be identified years afterwards. The importance of keeping tissues, sections, slides, &c., distinctly labelled cannot be too strongly impressed on the beginner. The name, date, and other particulars should be invariably written on the label at the time. At first the student will be inclined to neglect this, as he will recognize his pieces of tissue and sections so readily merely by their shape and general appearance. But as time elapses and similar specimens accumulate, he will find it most difficult or even impossible to identify one from the other.

A number of 1 oz. and 2 oz. stoppered bottles for staining reagents.

The stopper of these should be fitted with a rod. This is done by simply heating the lower end of the stopper and the upper end of a piece of glass rod of suitable length in a blow-pipe, until they are plastic, and then pressing them together.

Watch glasses.—At least a dozen watch-glasses, in which to perform the operations of staining, clarifying, &c. Those with a flat bottom should be employed as they are less easily upset than the others.

Plenty of filter papers.

Both coarse ones, for use in the manufacture of reagents, and small fine white ones (2 1/2 inch) for filtering the staining fluids immediately before using them, should be procured. Before using them a few drops of alcohol or distilled water should be placed in them to saturate the paper. This not only allows the fluid to pass through more rapidly, but prevents a portion of it being wasted through being absorbed by the pores of the paper.

Several needles mounted in handles.

They must be kept very bright and smooth, and care must be taken that the point does not get turned up.

A large and small funnel.

Several pipettes consisting of pieces of glass tube with an internal diameter of  1/8″ and about ten inches long, drawn out almost to a point at one end.

Section lifter.—This instrument is required for transferring sections from one reagent to another, or from oil of cloves, &c., to the slide. The most convenient form is Woodhead’s, made of thin sheet copper, which allows the blade to be bent at any angle to the stem. The stem or handle is about six inches long, and continuous with, and at an angle to it, a flat blade about  3/4 in. square with the corners rounded off. Larger ones can be obtained for mounting sections of large size, e.g., kidney, medulla oblongata, &c. The surface of the blade should be brightly polished, and kept scrupulously clean.

Ordinary dissecting forceps.

One or two scalpels.

A pair of fine scissors.

A razor or other instrument for cutting sections.

A smooth oil stone for keeping the razors and knives properly sharpened.

A spirit lamp for warming the staining fluids.

A few test tubes.

A minim measure.

Scales and small weights.

A gross of ground glass slides 3 x 1 in.

Half a gross of ground glass slides 3 x 1 1/2 in.

Half an ounce of thinnest coverslips,  7/8 in. diameter.

Quarter of an ounce of thinnest coverslips, 1 1/4 in. diameter.

Microscope.—This is not the place for a description of the microscope as an optical instrument, but some hints as to the selection of one may be found useful.

Showy microscopes with much brass work should be avoided, simplicity of construction being a great recommendation. The microscope should have a large heavy base, either of the horse-shoe or tripod pattern, large enough to afford a firm base when the microscope is tilted.

Mechanical stages are unnecessary and they add greatly to the expense, and very little to the utility of the instrument for ordinary histological work. Binocular arrangements also are of little use for this purpose.

The microscope should be provided with a coarse and fine adjustment, which should be most carefully tested before purchasing the instrument. They should work freely and smoothly, and the slightest turn in either direction should at once alter the focus.

There should be a reversible mirror, one side being concave and the other plane. The concave surface is the one usually employed, the plane surface being chiefly used in conjunction with the sub-stage condenser for the examination of micro-organisms. There should be an eye-piece of moderate magnifying power. Very powerful eye-pieces do not reveal additional details, but merely enlarge the image, and with it any defects that may be produced there by faults in the objective. Eye-pieces II. and IV. of most makers will be ample for most requirements.

Objectives.—These are the most important parts of the microscope, and the student will be well advised if he spends a little extra money to secure good lenses.

Most objectives and stands are now made with a universal thread, so that any objective will fit any make of stand. Many workers provide themselves with a cheap stand such as that supplied by Leitz, and then fit it with lenses by Zeiss, or other first class maker.

The most useful lenses are the 1 in. low power lens, and  1/5 in. or  1/6 in. high power, or No. 3 and No. 7 of Continental makers, or Zeiss’s A and D. A  1/2 in. lens will also be found very useful.

For minute work, such as bacteriology and blood investigations, higher powers will be required,  1/8 or  1/12 immersion lenses. These objectives come extremely close to the object, and very thin cover glasses must be employed. In order to avoid the refraction caused by the rays traversing the air between the coverslip and lens some immersion fluid is placed between the two. With some lenses water is employed, but usually an oil having the same refractive index as glass is used, and the one most generally employed is cedar oil (Zeiss prefers the oil from the species Juniperus virginiana). A spot of oil is placed with a rod just over the object to be examined and the objective carefully lowered by the coarse adjustment till it comes in contact with the droplet of oil. The focussing should then be managed with the fine adjustment only.

When the section has been examined the oil must be removed from the lens. For this purpose a soft silk handkerchief or a special piece of chamois leather may be employed, and used very gently. If all the oil cannot be removed, the handkerchief may be moistened with a little absolute alcohol, and the lens hastily wiped. The alcohol must not be allowed to remain in contact with the lens as it is a solvent of Canada balsam with which the lenses are often cemented in position.

Fig. 1.—Double or Triple Nose-​piece.

Double or triple nose-piece (fig. 1).—This mechanical arrangement is placed on the lower end of the tube. Two or three objectives of different magnifying power are attached to it. The nose-piece rotates round a central pivot in such a way that the objectives can successively be brought accurately into position above the object on the stage. It is, therefore, a moment’s work to replace a high power objective by a low power one and vice versa. It is an extremely convenient time-saving appliance, and by its use the risk of dropping and injuring the objectives when screwing them on and off frequently is avoided. Those whose microscopes are not already fitted with this appliance can easily have one fitted on at a cost of about a sovereign.

Substage condenser.—This mechanism for concentrating light on the object is a necessity for bacteriological work. The most convenient form is Abbe’s illuminating apparatus (fig. 2).

This consists of a system of short focus lenses which collects the light received by the mirror, and throws it on the object. The amount of light received from the mirror is controlled by an “iris diaphragm,” the aperture of which can be dilated or contracted by moving a small lever at the side. It can be fitted on to most microscope stands, but it is better to get a stand in the first instance which is constructed to carry one.

The cost of a microscope varies from two guineas to two hundred. There are many excellent microscopes in the market, and of these several may be mentioned which the writer has found to work satisfactorily.

Fig. 2.—Abbe’s Illuminating Apparatus.

Of the cheaper student’s microscopes the “Star” microscope made by Messrs. R. and J. Beck, of Cornhill, E.C., will be found a safe investment. It may be obtained with coarse and fine adjustment, nose-piece, and 1 in. and  1/4 in. objectives, for about £5. Those who require a better instrument will find Beck’s “Pathological” microscope fitted with nose-piece, Abbe’s illuminator, &c., for £16, meet all requirements.

Leitz of Jena, supplies two good and cheap microscopes for £3 10s. and £5. They are not, however, of uniform excellence, and they should be carefully tested by some competent judge before the purchase is completed. Leitz immersion lenses are cheap, and often extremely good, but should be carefully tested beforehand, as their quality is not quite uniform. The microscopes can be obtained from Mr. A. Frazer, Teviot Place, Edinburgh.

The “Bacteriological” microscope, made by Messrs. Swift, of Tottenham Court Road, is one with which no one can be disappointed. It is sold with Abbe’s condenser, triple nose-piece,  1/6 in., and a  1/12 in. immersion objective, for just under £20. Both stand and lenses are turned out in Swift’s first-class style, and those who can afford the initial outlay will not regret it. Or the stand may be purchased, and the objectives and accessories added singly from time to time.

Among Continental makers, excellent microscopes for histological work are turned out by many makers. Zeiss’s lenses stand deservedly high in reputation, as no faulty Zeiss lens ever leaves the works, and their optical properties are nearly perfect. For this guarantee, however, the purchaser has to pay somewhat higher prices, but the money is well invested. Zeiss’s agency is at 29 Margaret Street, Regent Street, W.

Reichert, of Vienna, sells microscopes and lenses which are modelled on the lines of those of Zeiss, and though cheaper are often equal to them in excellence, but the quality is not quite uniform. His instruments can be obtained through any optician, but his agent in this country is Mr. A. Frazer, Teviot Place, Edinburgh.

Before buying a microscope the student should obtain an illustrated price list from any of the firms mentioned above, and, having selected an instrument, he should test it very carefully, or better get some experienced friend to test it for him, before deciding to purchase it. Delicate test objects such as diatoms, scales of butterfly’s wing, or a stained specimen of micro-organisms should be employed. The coarse and fine adjustments should be tried. They should work freely and smoothly and without any delay. The definition of the lens must be tested with the fine objects mentioned. The field should be quite flat, i.e., every part should be in focus at the same time, and the definition should be perfectly sharp and accurate, and the test objects without double contour. The field should be totally free from prismatic colours. If there is a halo of colour around the objects it indicates a defect in the optical properties of the objective, and another should be selected.

A microscope must always be treated with the greatest care. Jars and falls tend to slightly loosen and shift the lenses, and to permanently impair its optical properties. Dust must be most carefully excluded. This is best effected by keeping the instrument under a glass bell jar when not in use. The lenses should be wiped as little as possible, and when it is necessary, very soft chamois leather should be employed. The microscope must be kept in a dry room, or the brass work will soon tarnish and the steel parts will tend to rust.

CHAPTER II.

Hardening Processes.

For the satisfactory examination of tissues it is necessary that they should be “hardened” in certain fluids. The object of this is to give the specimens greater consistence, so that thin sections may be more readily obtained and more safely manipulated, and also to “fix” the tissue element as far as possible in the same relative position as in the living body. The hardening process also acts on the protoplasm of the cells, and prevents their swelling up when placed in water, and in the various staining fluids.

The fluid used must be one which will not itself injure the specimen, and which can be thoroughly removed by washing, so that it may not interfere with staining operations. The specimens should be kept while hardening in wide mouthed bottles, on the bottom of which a little cotton wool or tow has been laid. This allows the hardening fluid to come freely in contact with the under surface of the pieces of tissue, and prevents their being flattened against the hard glass bottom.

The hardening fluid requires changing occasionally. This should always be done at the end of twenty-four hours, in order to get rid of any deposit of blood, &c., that may have accumulated. Besides this, the tissue when placed in the fluid contained a good deal of water which will have diluted it and consequently an early change is desirable. Afterwards the fluid requires to be changed only as often as it becomes turbid, or any deposit occurs, usually about once a week.

While hardening, specimens should be kept in a cool place, as warmth favours changes in the cells, &c.

In manipulating the portions of organs, forceps should always be used and these with great gentleness. The specimens should never be impaled with needles, or unsightly holes, which may even be mistaken for pathological appearances, will appear when a section is examined under the microscope.

It requires some practice to know when the tissue is sufficiently hardened. The object aimed at is to make them not really hard but tough. It is almost unnecessary to add that in testing this with the fingers the utmost gentleness must be observed, or serious damage may be done to the tissue.

When the tissue is sufficiently hardened the hardening fluid must be thoroughly dissolved out. This is most quickly effected by placing the specimen in a basin into which cold water from a tap is constantly running. The tissue may then be removed (forceps always being used and never the needle) and placed in an imbedding medium as subsequently directed; or, if it is not to be cut at once, into equal parts of methylated spirit and water, in which it may be kept indefinitely, the fluid being changed if it becomes at all cloudy.

It is unnecessary for ordinary work to have more than the following hardening fluids:—
Müller’s fluid:—

Two drachms of carbolic acid are sometimes added to each pint of the fluid but as a rule it is not necessary.

Müller’s fluid is the most generally useful of the various fluids employed, for the following reasons:—

1. It causes very little shrinking of the elements of the tissue, and hence may be employed for most delicate objects, e.g., the retina and embryos.

2. In consequence of its not making the tissues shrink, it does not squeeze the blood out of the vessels and where the organ has been congested before death, we may, by using Müller’s fluid, preserve a natural injection of the capillaries.

3. There is comparatively little danger of over-hardening the tissue and rendering it brittle.

4. Sections of organs hardened in Müller’s fluid are usually firm and easy to manipulate. They do not tend to curl up or adhere to one another as much as those hardened in spirit.

5. It readily permeates the tissues, and hence large portions of organs, or even the entire organ may be satisfactorily hardened in it.

6. It is very cheap. A gallon can be made up for about eightpence.

The fluid has however certain slight drawbacks:—

1. The hardening process is a slow one occupying four to eight weeks.

2. The fluid gives a permanent dingy colour to the tissue. This does not cause any inconvenience for microscopic purposes, but it is a disadvantage when it is intended to preserve the rest of the specimen, as a naked eye preparation. In such cases the organ should be hardened in spirit, carbolic acid, or formal.

Müller’s fluid can be used for almost any tissue. It is especially useful for those which contain a large quantity of fluid, or of blood, and is essential for nerve tissues which it is intended to stain by Pal’s method (p. [89]).

To harden a specimen in it at least twenty times the bulk of fluid must be employed.

The fluid must be changed on the third day, and afterwards about every week as may be required.

Methylated spirit is a very useful hardening agent. It hardens in one to three weeks according to the size of the tissue and the quantity of spirit used. Its disadvantages are:—

1. It is more apt to overharden than Müller’s fluid.

2. It causes a great deal of shrinking of the tissue and thus squeezes much of the blood out of the vessels.

It is most useful in hardening tissues containing much epithelium, e.g., kidney, epithelioma, &c.

Spirit is also frequently employed to complete the hardening by Müller’s fluid and to preserve tissues after they have been hardened.

About ten or fifteen times the bulk of spirit should be used for one of the tissues. The fluid should be changed on the third day and afterwards as required.

Müller’s fluid and spirit.—This is a useful combination for many purposes. It is made thus:—Müller’s fluid, three parts; methylated spirit, one part.

The fluid must be allowed to cool after mixing before being used, and if necessary filtered. It will harden specimens satisfactorily in three weeks.

Müller’s fluid and formal.—Is an extremely useful mixture made by adding one part of formal to nine of Müller’s fluid. It hardens in a much shorter time than Müller’s fluid and causes very little shrinkage.

Absolute alcohol.—Used as a hardening agent where the tissues are to be examined for micro-organisms, and for specimens to be stained by Nissl’s method (p. [101]). A cheaper and equally effective hardening medium is made by dehydrating methylated spirit by adding one ounce of fused carbonate of potassium to each pint of methylated spirit, and decanting.

Small pieces must be used. The depths of the block should not exceed  3/8 inch. The fluid should be changed on the third day. Hardening will be completed in about ten days or even earlier.

Osmic acid.—For rapidity of action, and for rapid fixing of all the tissue elements in their natural position osmic acid is one of the best hardening reagents we possess.

Its disadvantages as a hardening agent are:—

1. Its expense.

2. Its irritating and corrosive vapour.

3. The fact that only small pieces of tissue can be hardened in it, since the external surface is very rapidly hardened and thus the fluid is prevented from penetrating into the centre of the lump.

It is most frequently used as a hardening agent for very delicate structures, such as the retina, or embryos, or for fresh sections of brain (p. [95]).

The acid itself may be procured in sealed tubes, each containing one gramme. These should be broken in a bottle under sufficient distilled water to make a one per cent. solution. The bottle containing it should be covered with brown paper to exclude the light. For hardening purposes small pieces of the tissue, not much larger than a pea, should be placed in the acid, the one per cent. solution being diluted with five to ten volumes of distilled water. The tissues may be left in this for from three to five days. They must then be thoroughly washed in distilled water and may afterwards be preserved in methylated spirit.

Both the hardening and the subsequent washing must be carried on in the dark.

Osmic acid is also a most valuable staining reagent (see p. [81]).

Carbolic acid (5 per cent.).—May be used to harden almost any tissue, but is particularly useful for hardening nervous tissues such as brain or spinal cord which are afterwards to be preserved as museum specimens. It does not discharge the colour of a specimen so rapidly as spirit.

Three or four times the bulk of fluid should be used. It requires changing at the end of twenty-four hours, and again at the end of the first week.

Saturated aqueous solution of corrosive sublimate is one of the most convenient hardening reagents for small pieces of delicate tissue, e.g., embryos. It hardens them in a few days. When they are sufficiently hardened the mercurial salt should be removed by washing first in methylated spirit for a few hours and then in running water.

Formal.—An aqueous solution containing about 35 per cent. of formaldehyde. It is a rapid hardening agent, causes very little shrinkage of the tissues, and does not discharge the colour of the specimens as much as alcohol. For hardening formal should be used as a two to five per cent. solution in distilled water. It may also be used as a ten per cent. solution for mounting museum preparations, but there is some tendency for a cloudy deposit to form on the glass after a time. It is the most suitable hardening agent at our disposal for eyes. It rapidly fixes the tissue elements, but does not cause much contraction. It may also be used for hardening the brain and spinal cord. Large quantities of fluid must be used for the latter purpose and it must be frequently changed. As soon as they are sufficiently hardened they should be transferred to methylated spirit.

Marchi’s fluid.—This consists of:—

It is used for hardening specimens as a preliminary to Golgi’s method for staining nerve cells (p. [97]), and also to complete the hardening of sections of spinal cord, &c., before employing Schäfer’s modification of the Weigert Pal hæmatoxyline method (p. [91]).

It is also used as a stain for recently degenerated nerve tracts and fibres, especially after experimental lesions.

The fluid has little penetrating power, and therefore tissues must be cut into small pieces, about  3/8 inch cube. It is not necessary to place them in this fluid at once on removal from the body, but the preliminary hardening must be in Müller’s fluid and not in alcohol, &c.

Special Hardening Reagents for Rapid Fixation in Order to Study Cell Structure.

1. Alcohol.

2. Flemming’s solution (modified by Friedmann):—

Small pieces should be hardened in this fluid for twelve to twenty-four hours, and then washed and transferred to alcohol for some days before staining.

3. Nitric acid.—A ten per cent. solution in distilled water. It hardens the tissue in three to four hours, and should be followed by 70 per cent. alcohol, the hardening being completed in absolute alcohol.

In using any of these methods it is necessary that the tissue be removed from the body during life or immediately after death. They are employed for revealing the changes in the cells and their nuclei in rapidly growing or inflamed tissue, for studying karyokinesis in cancer cells, and investigating the appearance of nerve cells and gland cells at rest, when actively employed and when fatigued; and they are also most useful in preparing specimens of the “parasitic bodies” which have been described in many cancer cells.

Decalcifying Fluids.

Used in the preparation of bone, tooth, osseous tumours, &c. The two best fluids for general use are:—

Chromic and nitric fluid.—This is made as follows:—

If the bone is not very compact the fluid may be used diluted with an equal quantity of water. A large quantity of fluid should be used, and like all decalcifying fluids, it should be frequently changed.

As soon as the specimen is sufficiently flexible, it should be thoroughly washed in running water for some hours, and then transferred to spirit until it is convenient to cut sections.

Von Ebner’s solution:—

It is a very useful decalcifying agent, but causes the fibrous elements to swell up rather more than chromic and nitric fluid. A large quantity of the fluid must be used, and it should be changed daily. It must be very thoroughly washed out in running water when the decalcification is completed.

Bleaching solution (eau de Javelle).

Shake up well.

Mix the two solutions. Allow them to stand for an hour and filter.

It is used chiefly for clearing vegetable sections but may also be used for sections containing a large quantity of pigment. It is particularly useful in decolourizing sections of “madura foot” due to the presence of a black fungus.

CHAPTER III.

Section Cutting.

Embedding of sections.—Before sections are made the tissues require to be embedded in some fluid, which will permeate their interstices, and is capable of being rendered firm so as to support the most delicate parts when the knife passes through the tissue.

The most generally useful substances are:—

(1) gum, (2) celloidin, (3) paraffin or wax.

Gum.—Picked colourless gum arabic 2 parts, cold water 3 parts.

Leave with frequent stirring until dissolved. Add ten drops of carbolic acid to each ounce of the mucilage.

Specimens are thoroughly freed from all trace of the hardening fluid by washing in water, and the tissue is then placed in the gum solution for at least twelve hours, or if enough carbolic acid be added, it may be left there for an indefinite time.

When frozen, gum forms a firm non-crystalline mass, which supports the tissue on all sides. It must not be frozen too deeply, or it becomes hard and rather brittle and is apt to injure the razor. If this have occurred the surface can be softened sufficiently by breathing gently on it.

After cutting in gum, the sections are gently removed from the knife into distilled water by a soft camel’s hair brush, and left there for an hour or two, until the medium is entirely dissolved out. They may then be stained and mounted, or they may be put away in spirit for an indefinite time, and then stained and mounted.

Celloidin is for many purposes almost an ideal embedding medium. (1) It has great penetrating power; (2) it can be made of an admirable consistence for cutting purposes; (3) after sections are made it allows them to be very freely manipulated without fear of injuring them: (4) and being perfectly transparent and homogeneous in thin sections, it does not require to be removed from a section before mounting. It is insoluble in water, and in weak spirit; slightly soluble in alcohol of more than 90 per cent. strength, and very readily soluble in ether, or in a mixture of alcohol and ether. The last solvent is the one commonly employed.

The embedding solution is made thus:—

Take some pure celloidin (“Schering’s,” sold in boxes containing an ounce of shavings, is very good) and pour on it about eight times its volume of a mixture of equal parts of absolute alcohol and ether. Allow this to stand all night until the celloidin is dissolved. The solution should be made about the consistence of ordinary mucilage.

It is also convenient to have a thinner solution made by using double the proportion of alcohol and ether. Both solutions should be kept in wide mouthed stoppered bottles, and the stopper should be well greased with vaseline as an additional obstacle to the evaporation of the volatile ether.

Before embedding a specimen it is necessary to dehydrate it thoroughly for twelve to twenty-four hours in absolute alcohol. It should then be placed in a mixture containing alcohol and ether for an hour or two, and afterwards transferred to the thin solution of celloidin for twenty-four hours, and then to the thick solution for the same period. The celloidin penetrates slowly and in the case of nerve tissues and other delicate structures it is wise to give the full allowance of time for the different steps. When the tissue has been thoroughly permeated by the celloidin, it is gently removed from the celloidin and placed in position on a piece of cork of suitable size for clamping in the holder of the microtome. Celloidin is painted round the object so that it is supported on every side. It is then left exposed to the air until the surface has become firm, when the cork is placed, with the tissue downwards, in methylated spirit. The cork floats but the tissue and celloidin remain submerged. At the end of twenty-four hours the celloidin will have become semi-opaque and opalescent, and of the same consistence as hard boiled white of egg. When it is impossible to wait so long, rapid hardening of the celloidin may be secured by immersing it in methylated chloroform in place of spirit, but the slower method gives more uniformly satisfactory results.

Pieces of tissue embedded in celloidin may also be cut on a freezing microtome. After the celloidin has become firm by immersion in methylated spirit, the tissue with the celloidin round it may be cut off the cork, washed in water to remove the alcohol, and then soaked for an hour or two in gum, placed on the plate of an ether spray microtome, frozen and cut in the usual way.

Subsequent staining operations are conducted in the same way as for sections cut by hand or in gum. As celloidin is only slightly stained by hæmatoxylin, alum carmine, borax carmine, &c., it is not necessary to remove it from the sections, but it exhibits so intense a staining reaction with aniline dyes that it is necessary to remove it by treatment with alcohol and ether either before or after the staining operation.

The sections after staining may be mounted in Farrant’s solution (p. [59]), or in Canada balsam (p. [61]). If the latter medium is employed, the section should be clarified, after dehydration in alcohol, by means of oil of bergamot, or oil of origanum, instead of oil of cloves, as the latter dissolves out the celloidin and causes the section to break up.

Celloidin is most useful for cutting sections of the coats of the eye, of the internal ear, and of bone marrow. It should always be used for the Weigert-Pal hæmatoxyline method of staining the nervous centres, as it protects the section from being injured by the transference from one fluid to another which is repeatedly required during the process. The stain is completely discharged from the celloidin by the decolourising solution used (p. [90]).

Paraffin.—Paraffin is a very convenient embedding medium for delicate structures, as very thin sections can be obtained and the paraffin need not be removed from the section until the latter is safely on the slide. It is unsuitable for large sections. Staining operations are not easily carried out after cutting in paraffin, and it is better to stain the blocks of tissue in bulk before embedding. The best stains for penetrating are borax carmine (p. [75]), alum carmine (p. [76]), and Kleinenberg’s hæmatoxyline (p. [70]). The tissue must be left in them for four to ten days.

Various kinds of paraffin are employed. It is usual to keep two kinds, one “soft,” melting at 110° F., and another “hard,” melting at 140° F. A mixture of two parts of the hard and one of the soft will be found most generally useful. In winter a large proportion of the soft variety and in hot weather a larger proportion of the hard may be required. A paraffin mass which is always available has been suggested recently by Dr. F. E. Batten, who employs an ordinary white candle, composed of paraffin and wax. If the mass is found to be too hard, it can easily be made of a suitable consistence by adding a little paraffin with a low melting point.

To prepare a piece of tissue for embedding in paraffin, it should be stained, washed in distilled water, and as much moisture as possible removed by blotting paper. The block is then dehydrated, first in methylated spirit for several hours, finally in absolute alcohol. It is taken carefully by means of forceps from the alcohol and placed in xylol for an hour or two according to size. Superfluous xylol is removed from the surface, and the tissue placed in the melted paraffin. This will set round the cold tissue at once, but soon melts again and must be kept at a temperature just above melting point for one to four hours, according to size. The tissue is then transferred to a mould (which can be easily made of paper), about half an inch cube, and melted paraffin poured round it until the mould is full. The mould may be made by folding a piece of paper to form a box about half an inch cube, or a small pill box may be used. Another convenient method is to place two L-shaped pieces of lead in contact with each other so as to enclose a space of suitable size as in the diagram (fig. [3]). The tissue is now hermetically sealed, and can be kept indefinitely if it is not convenient to cut it at the time. To prepare it for cutting, all superfluous paraffin is trimmed away with a warm knife, and the block is fixed on a piece of wood, cut so as to suit the clamp of the microtome, by melting the lower end of the paraffin block with a hot needle or wire and pressing it down on the wood.

Fig. 3.

When sections are cut they may be transferred singly to the slide (which should be lightly smeared beforehand with a saturated solution of celloidin in oil of cloves), or they may be cut so that the back of one section of the paraffin block adheres to the front of the next, and in this way a continuous delicate ribbon of serial sections is obtained. The ribbon is broken up into lengths of about two and a half inches and transferred to the slide, on which several ribbons may be placed side by side, and so a large number of sections kept in the order in which they are cut. A mark should be made on the slide to indicate where the series begins, and each slide should be numbered, so that the exact position of each section in the series can be recognised at once.

Before mounting, the paraffin must be removed from the sections. This is easily done on the slide in the case of single sections and of ribbons. If the sections are curled, a little warmth will make them unbend and lie flat. The slide is warmed over a spirit lamp until the paraffin just melts. The sections will keep their places owing to the celloidin beneath. Xylol is then allowed to flow over the slide from a pipette, until the paraffin has been completely dissolved, which can be ascertained by glancing at the sections under the low power of the microscope. The slide is placed in an almost vertical position to let the xylol drain off, excess is wiped off from the edge of the slide with blotting paper, a drop of Canada balsam solution (p. [61]) is run on the slide, and a cover-glass of suitable size is applied.

Microtomes.—After a large amount of practice, persons with a fair amount of manual dexterity may acquire sufficient skill to be able to cut very satisfactory sections of specimens embedded in paraffin, &c., by hand. In the Pathological Laboratory of a large German University, until quite recently the use of a microtome was prohibited by the Professor, who is himself a most distinguished histologist. The amount of time expended before one acquires the necessary skill, and the cheapness and great convenience of the modern microtome have combined to throw hand cutting into the background, and some form of microtome is now almost universally adopted.

Fig. 4.—Cathcart’s Ether Spray Microtome.

A, B. Wooden frame and supports. C. Glass runners. G. Screw for raising the zinc plate H. J. Ether bottle. L. Tube from air bellows.

Of these there are a very large number in the market, each having special advantages, and often special drawbacks. A few of the more generally useful only will be described. We have microtomes for cutting in gum frozen by ether spray or ice, and those intended for cutting in paraffin or celloidin.

Cathcart’s ether spray microtome (fig. 4).—This, or its more recent modifications (see later), is perhaps the most useful and economical microtome for the purposes of the student. Its prime cost is low, it is small and portable as well as being clean and inexpensive to work with.

It consists of an oak frame which can be firmly clamped on to a table. On this frame are two narrow parallel supports about two inches high, which are covered by strips of plate glass, and serve as smooth rests along which the razor may glide in making sections. Between them is a brass well and in this a zinc plate firmly fixed in the horizontal position, which is almost at the level of the glass runners. It is capable of being raised or lowered through about  3/8 inch by means of a screw with a very fine and accurate thread. This screw is turned by a large milled wheel beneath the microtome. Just beneath the zinc plate are two small tubes, one connected with an india-rubber bellows, the other with a bottle at the side which contains ether. As the air issues from the first tube, it passes over the open end of the second, and thus draws the ether out and makes it play on the zinc plate, and at the same time causes it rapidly to evaporate, and so reduces the temperature of the zinc plate.

In cutting sections with this microtome the tissue is taken out of the gum and placed on the zinc plate. The bellows are then worked until the gum on the zinc plate is completely frozen. The plate should be lowered by means of the screw until the surface of the piece of tissue is on a level with the glass runners. These and the razor should then be wetted with water. The razor being held firmly in the hand is pushed along the glass runners in a rather oblique direction. The plate should then be raised by turning the screw below through a very small arc, another section taken off and so on. Sections are carefully removed from the razor to a vessel of water by means of a soft wet camel’s hair brush. The needle should never be used for this purpose.

If the specimen is very delicate, and likely to be spoiled by being curled up on the knife, the latter should be kept cold by frequently dipping it in a vessel containing lumps of ice in water. The gum will then remain frozen after cutting, and support the tissue better. Each section should be at once transferred to a glass slide from the knife, washing it off with a stream of ice water from a pipette.

The knife that is used may be an ordinary razor, with the edge ground straight. It requires to be held steadily with both hands. As this is rather inconvenient, Dr. Sheridan Délépine suggested the employment of an ordinary plane iron such as is used in a carpenter’s plane. This only requires one hand, and the other can be kept on the head of the screw beneath to raise the plate at once after each stroke of the knife. Its disadvantages are that it is rather heavy for prolonged working, and that it is less easy to “set” than a razor.

A. Frazer has recently introduced a valuable improvement in the Cathcart microtome (fig. [5]).

In this the brass frame carrying the zinc plate and ether spray tubes is surrounded by a brass cylinder, in which it fits accurately, and is pushed up as desired by turning the screw beneath the instrument. This brass frame and with it the zinc plate, &c., can be easily drawn altogether out of the outer tube, and replaced by a second brass well, which exactly fits its place and can be raised by the screw as desired. In this is a small toothed clamp which can be screwed up so as to hold a piece of wood carrying a piece of tissue embedded in paraffin. Sections can also be cut in celloidin with this instrument, but as oblique strokes with the knife cannot be made, it is impossible to get very thin sections. The combined microtome can be obtained for a guinea from Frazer, 22 Teviot Row, Edinburgh.

Fig. 5.—Frazer’s Modification of Cathcart’s Microtome.

A. Microtome arranged for ether spray. B. Cylinder with clamp for holding object embedded in celloidin, &c. to replace ether spray apparatus.

There is another modification which is more generally useful, and at the same time more expensive than the original model. In this, instead of glass runners to support the knife, there is a flat glass plate about eight inches square sufficiently large to allow of “Swift’s plough” (fig. [6]) being used for the purpose of cutting sections. This instrument consists of a triangular brass frame, supported on three legs, each of which is a screw, tipped with ivory. There is one screw in front and two behind. Beneath the plate, and held in position by the posterior screws in front, and a little clamp behind, is a razor with the edge directed forwards. The edge can be raised or depressed by turning the anterior screw, on which the frame is supported. Before sections are cut the edge of the razor should be brought down to the level of the tissue, taking care that all the legs are equal in length. The plough should then be firmly grasped with both hands, (the index finger of one hand being left free to turn the anterior screw) and pushed rather obliquely through the tissue. The edge of the razor is then slightly lowered by turning the screw through a very small angle, and another section made, and so on. With a little practice very thin uniform sections may be made with great rapidity.

Another useful ether spray microtome is that made by Jung of Heidelburg. The knife swings round a pivot, and there is an ingenious ratchet arrangement which works synchronously with each swing of the knife, to raise the tissue automatically the requisite distance for the next section to be made. The exact thickness of the sections can be graduated with great nicety by a simple contrivance. The instrument can be obtained in this country for about £2. It works satisfactorily, but, with practice, the student will get equally good results with the cheaper “Cathcart.”

Williams’ ice freezing microtome (fig. 6).

This consists of a round mahogany water-tight box provided with an exit tube below, and covered with a strong plate glass lid. Firmly fixed in the centre of the floor of the box is a stout brass pillar surmounted by a brass disc which fits into a hole in the centre of the glass lid, so that its surface is on a level with that of the lid.

Fig. 6.—Williams’ Ice Freezing Microtome, with Swift’s plough.

To use it, the box is filled with alternate layers of pounded ice and salt; the lid is then put on and fixed by means of a lateral screw. The tissue to be frozen is gently removed from the gum and placed on the brass disc and plenty of gum painted round it. It should then be covered with a tin cap for a few minutes until frozen. Sections are made with a Swift’s Plough (p. [44]).

Fig. 7.—Schanze Microtome (see text).

Schanze microtome (fig. 7) is the pattern used in the Leipsic laboratories. It consists of a heavy iron frame with a large base. The knife is carried in a clamp which slides along the full length of the instrument, gliding upon two smooth plates of iron which are arranged at an angle to one another. The knife must be moved very steadily and gently, as when using a long blade vibrations are easily set up which prevent good sections being obtained. The surfaces of contact must be kept scrupulously free from dust, and lubricated with equal parts of olive oil and castor oil. There are several object holders, which can be removed and interchanged, one connected with an ether spray apparatus, another suitable for holding an object embedded in paraffin, and a third for grasping an object embedded in celloidin. When celloidin is employed, a specially long knife must be used, and it must be fixed very obliquely in the clamp. The object holder is raised by a fine screw worked by a large brass toothed wheel. There is a ratchet arrangement, by which the object may be raised automatically any desired distance, after each stroke of the knife. It gives most satisfactory results with celloidin and paraffin. (Messrs. R. and J. Beck are the agents). Its cost is about £5.

Becker’s microtome is made on exactly the same principles as the Schanze. The modifications are that the carrier glides on glass plates instead of iron ones, and that instead of the whole surface of the carrier being in contact with the plates, there are a few smooth ivory buttons only. Friction is thus reduced to a minimum, and very uniform sections can be obtained. The price is the same as that of the Schanze.

Frazer has introduced a “student’s sliding microtome” on the same principle as the Schanze which costs about £3.

The Cambridge Rocking microtome.—This instrument, as made by the Cambridge Scientific Instrument Company, or the slightly modified form made by Messrs. Swift (fig. [8]), is the best instrument for cutting sections of small objects embedded in paraffin. Ribbons of serial sections can be obtained from it with greater ease and certainty than with other microtomes. This microtome differs from those which have been previously described in that the knife is fixed, while the object is moveable. The microtome consists of an oblong heavy metal stand. A long bar is arranged so that it rides in see-saw fashion on two strong vertical pillars arising from the frame. One end of this bar is hollow, and receives the piece of wood carrying the tissue embedded in paraffin, which is firmly clamped in position. This end is depressed by means of a strong spiral spring. In order to raise it there is an arrangement by which the other end of the bar is depressed by a cord which revolves round a pulley. When the handle is turned, the tissue is raised, and when the cord is relaxed, the spring pulls the tissue firmly and steadily down. The razor, which must have a straight edge, is fixed firmly by screws, with its edge upwards at the end of the microtome. The object is then adjusted so that in its descent a thin slice is taken off by the razor. There is an ingenious arrangement by which the depression of the bar to raise the section pushes it a little further in the direction of the razor. The distance can be graduated from  1/500 to  1/3000 inch. The actual working of the machine is therefore very simple. The position of the block containing the tissue to be cut having been adjusted so that the razor just cuts it, the free end is depressed by means of the pulley. This also pushes the section a little beyond the razor. The strong spring then draws the tissue steadily past the edge of the razor, and a thin section is left on the blade. This may be at once transferred to a slide, or if the paraffin be of the proper consistence, another cut may be made, when the two sections should adhere by their edges, and so by repeating the movement a continuous ribbon may be obtained. If there is difficulty in obtaining a good ribbon, it will usually be got over by taking a little soft paraffin and attaching it by means of a hot needle to the lower end of the paraffin block. The cost of the instrument is about £5.

Fig. 8.—Swift’s Modification of the Cambridge Rocking Microtome.

Fresh sections.—Although these are not so satisfactory as hardened specimens for accurate histological work, it is often very useful to make them both in the post-mortem room where an immediate opinion of the nature of the tumour or diseased organ is desired, and also in the operating theatre. With a little practice sections may be cut, stained, and mounted, within ten minutes of the removal of the specimen from the body. In this way important information may be afforded to the operating surgeon, and in not a few cases it has caused the proposed treatment to be entirely altered. Thus, in one case, a supposed chronic periostitis was shown to be a sarcoma, and the limb was amputated. In another, a supposed sarcoma of the thigh was found to be a gumma, when a portion was removed and microscopically examined.

A portion of the specimen should be placed without any preparation on the zinc plate of the freezing microtome, and some gum painted round it. It is then frozen. The serum in the tissues is not in sufficient mass to injure the knife when it is frozen. The knife should be wetted with, and sections transferred to, either pericardial serum, or  3/4 per cent. solution (70 grains to the pint), of common salt, neither of which causes the cells to swell up as plain water does. They should be carefully floated out on a glass slide, an operation which requires much more patience than in the case of hardened sections, as fresh sections are less coherent and also more sticky, so that the edges tend to curl up on the knife, &c. They should then be examined, one unstained, simply mounted in salt solution; another stained with picrocarmine and examined in the saline solution; and a third stained in picrocarmine, mounted in Farrant’s solution, and preserved. The last usually gives the best results, the picrocarmine staining becoming quite brilliant after a week. The glycerine, however, is apt to make the sections shrink a good deal, and the weight of the cover-glass tends to break up the unhardened section.

CHAPTER IV.

Section Mounting.

1. By flotation.

In this method the section whether stained or unstained is placed in a bowl of water, or normal salt solution (p. [53]). A clean slide is then introduced into the water at an angle of about 60°, a little more than half of its length being submerged. The section is then brought up by the needle and floated as far as possible into position on the slide. One corner is then fixed by the needle, and on gently withdrawing the slide the section should lie flat. If any folds are left no attempt should be made to smoothen them out with a needle, but the slide should be re-immersed until the folded part of the section is under water. It should then be gently withdrawn, when the fold will disappear. This manœuvre must be repeated in different directions until the section lies quite smoothly on the slide. Stained and unstained sections are floated out in this way before being mounted in Farrant’s medium, and unstained sections previous to staining in picrocarmine.

2. By transference with a section lifter.

This method is employed in mounting in Canada balsam in order to transfer the section from the clarifying agent (p. [63]) to the slide. The lifter is polished, and insinuated under the section. The section being held in position by the needle is now raised from the fluid, excess of which is removed by holding the section in position with a mounted needle, and tilting the lifter so as to allow it to drain off.

Removal of air bubbles from sections.—When sections contain many air bubbles, the best plan is to leave them in methylated spirit for a time. The bubbles then coalesce and escape from the section.

For delicate structures and for fresh sections the transference to spirit, and the subsequent flying out of the section when returned to water are risky, and the best method of treating these is to put the vessel containing them under the receiver of an air pump, if one is available, and slightly exhausting the air.

The most frequent cause of air bubbles in mounted specimens, however, is the employment of cover-glasses which have not been thoroughly cleansed. Proper cleansing is best effected by placing the covers when bought in a shallow wide mouthed stoppered bottle containing strong nitric acid, and leaving them in this fluid for twenty-four hours. The acid should then be drained off and water run through the vessel from a tap, until the washings no longer give an acid reaction with litmus paper. The water should then be drained off, and the glasses covered with absolute alcohol. They can be removed one by one and rapidly dried as required. With cover-glasses properly cleansed in this manner, not only will air bubbles be avoided, but the covers will be dried much more easily with the cloth, and fewer will be broken in the process.

Another very frequent cause is the transference of air bubbles with the mounting medium on the glass rod. This occurs especially if the rod be fused to the stopper. The proper bottles to use, both for Farrant’s medium and balsam are “balsam bottles” which have no stopper, but the mouth is closed by a glass cap which fits accurately (fig. 9). A short glass rod is attached to the cap, and is used to transfer the medium to the slide.

Fig. 9.—Balsam bottle.

Treatment of folded sections.—The folding may be due:—

(1.) To the section having creased through being cut with a knife whose surface was not perfectly smooth. This is best remedied by placing the section in methylated spirit for a minute, and then transferring it to a bowl of clean water, when the section will rapidly rise to the top, and spread itself out flat on the surface of the water, in consequence of the alcohol rapidly diffusing out at the edges into the surrounding water.

(2) To the section containing a large amount of fat, as in those of the skin and subcutaneous tissue. The fat may be removed from the fat cells without materially altering the appearance of the section. This is done by dehydrating the section in alcohol, and then transferring to a watch glass containing ether or chloroform to extract the fat. The tissue should be washed free from ether in the alcohol and then transferred to the bowl of water, and allowed to float out. This process does not interfere with subsequent staining operations.

Mounting Media.

Farrant’s solution:—

Gum Arabic (picked, colourless)		equal parts.
Glycerine
Water

In making this solution the best gum arabic must be used, and only the clearest pieces of this. “Powdered gum acacia” should be avoided, as though it looks white it often yields a brown mucilage, and besides is frequently adulterated with starch, &c.

The glycerine and water should be mixed and the gum arabic added. The mixture should be allowed to stand for some weeks, with frequent stirring until the whole of the gum is dissolved. Then allow it to stand for a week or two longer in order that the dirt may subside, and the bubbles rise to the top. The scum should be removed and the clear fluid decanted from the sediment into a “Balsam bottle” (p. [58]) containing a few drops of a saturated solution of arseniate of sodium and a small lump of camphor.

If properly made it is an extremely useful mounting reagent. It does not clarify the tissues too much, and in consequence of its containing gum it dries at the edges and cements the cover-glass more or less firmly in a week or two. If this is not the case the medium contains too much glycerine and more gum must be added to compensate for this. This drying at the edge prevents any further evaporation while the glycerine keeps the section permanently moist.

The camphor and arseniate of sodium prevent the formation of fungi. Sections preserve their original appearance in this medium for many years. After a long time they are apt to become a little cloudy and granular.

Unstained sections should always be mounted in Farrant’s medium, as the Canada balsam process renders them quite transparent. It is suitable for almost any tissue stained or unstained, but sections of the nervous centres require to be mounted in Canada balsam, owing to the opacity of myelin when mounted in glycerine.

Canada balsam solution:—The medium is made thus:—

The ordinary Canada balsam which is of a treacly consistence is heated gently in a water bath for some hours, to drive off turpentine and other volatile oils. It is then allowed to cool to a yellow vitreous mass. Take of

Dried Canada balsam		equal parts.
Xylol

Leave till dissolved, stirring occasionally.

Unless the solution be perfectly clear, it must be filtered through a very thin paper, previously wetted with xylol. If the medium be too thick more xylol should be added, if too thin, the xylol should be allowed to evaporate until the medium is of the consistence of a thin syrup.

If the medium is made too thin much annoyance will be caused by its evaporating at the edge of the cover-glass, leaving an air-space, which will increase daily until the section is left quite dry. This should be remedied by putting another drop of balsam at the edge of the coverslip and allowing it to run in and displace the air. A ring of cement should be put on as early as possible afterwards.

The bottle in which the balsam is preserved must be very carefully dried before being filled and then rinsed out with absolute alcohol, and afterwards with xylol. Turpentine or benzol are often used instead of xylol in the preparation of the medium, and in the same proportion, but the latter is less apt to dissolve out the aniline colours from the sections.

To mount sections in Canada balsam they must be transferred first to a watch glass containing absolute alcohol or an alcoholic solution of some staining reagent, e.g., eosine (p. [72]) and left in it, no attempt being made to spread it out, until it is perfectly dehydrated, i.e., in about two minutes. It should then be transferred to the clarifying oil on a mounted needle, or on a section lifter, which must be perfectly dry as any spot of moisture that gets on to the section will resist the clarifying action of the oil, and will cause unsightly opaque areas when the section is mounted. Even breathing on the section on its way to the clarifying agent will prevent uniformity of clearing. Should white spots appear in the section while in the oil it must be taken out with as little oil as possible, and again dehydrated in absolute alcohol.

The process of clarifying must be performed in some medium in which Canada balsam is readily soluble, and which is also readily miscible with alcohol. Those most frequently employed are oil of cloves, xylol, oil of bergamot, oil of cedar, and origanum oil. The first named has always been much used because of its agreeable odour, its cheapness, and the ease with which it can be obtained. But it has the disadvantage of dissolving out many important staining reagents, especially eosine and the various aniline colours. In addition as it dissolves celloidin, sections cut in this medium tend to fall to pieces when transferred to oil of cloves, and one of the other oils (which have no solvent action on celloidin) should always be employed with celloidin sections. Oil of bergamot is the most generally useful, but rather expensive. Where there are special reasons for employing other dehydrating agents, they will be indicated in the special directions for particular staining methods in Chapters [VI]. and [VII].

As soon as the section is plunged into the oil, the alcohol rapidly diffuses out, so that the edges of the section fly out with it, and the section floats quite flat on the surface of the oil. When it is completely clarified (in about a minute), as shown by its sinking in the oil, it should be transferred to the slide by the section lifter, and the oil drained off. Excess of oil may be removed by pressing gently on the section with a flat piece of filter paper folded several times. If carefully performed this manœuvre will not injure the section, but it requires practice.

If the tissue is very delicate, and likely to be injured by changing from one vessel to another, or if it is larger than the section lifter will conveniently carry, it should be floated out on a glass slide, and, as much water as possible having been removed by blotting paper, should be dehydrated by adding a little alcohol from a pipette once or twice. Most of the alcohol should then be removed by tilting the slide, and before the remainder has evaporated, some oil of cloves or bergamot should be added from another pipette. The section will float on the oil at first, but the latter will gradually come through and appear on the top of the section. When this occurs the clarification is complete, and the oil may be run off by tilting the slide and the section mounted in Canada balsam.

Cementing of cover-glasses.—The cover-glasses may be cemented down to prevent their shifting and spoiling the specimen. If the cover-glass be circular, a Shadbolt’s turntable should be used. It consists simply of a horizontal heavy brass disc, rotating easily on a pivot. There are a number of circles traced on the disc concentrically. The slide is then fixed on the disc by means of the clips, so that the circumference of the cover-glass corresponds to one of the circles. The disc is then rotated and the cement applied to the edge of the cover-glass with a brush.

Many materials are employed. The most suitable are:—(1) Canada balsam, which is almost colourless and transparent and looks very neat. (2) Gold size. (3) Marine glue.

When these are dry a finished appearance may be given to the slide by laying on a ring of zinc white. This is made as follows:—

Oxide of zinc		1/2 drachm.
Benzole		half an ounce  of each.
Gum dammar

Preservation of sections.—They should be kept flat, and preserved from both light and dust. Very useful cardboard trays are now sold by almost all dealers in boxes made to contain twenty-four dozen slides for about eight shillings, or suitable cabinets may be constructed by a carpenter.

CHAPTER V.

General Staining Methods.

Much information may be obtained from unstained sections, and in most cases one section should be examined unstained, but the specimens mounted in this way are so transparent that it is difficult to study the details of the tissue. They are therefore usually prepared by treating them with some staining reagent, not merely to render them less transparent, but also to “differentiate” the elements of the section, by staining one part more deeply than another, or of a different colour. Thus hæmatoxyline stains the nuclei and rapidly growing parts of the tissue, leaving the formed material, as a rule, much more lightly tinted. Methyl violet again stains healthy tissues blue, and parts affected with waxy degeneration a red-violet colour. By combining stains also much differentiation of the tissue elements may be obtained. Sections should be stained with several reagents, as their effect on individual specimens varies a good deal.

The following are the most useful stains for general purposes:—

Logwood.—This or its purified principle hæmatoxyline is the most useful general stain. The hæmatoxyline itself is preferable, giving more constant results, and less diffuse staining.

For general staining purposes the following formula will be found to give excellent results:—
Hæmatoxyline. Schuchardt’s formula.—

Add (a) to (b) drop by drop and with constant agitation. Keep for some days exposed to diffuse daylight until its colour is so deep that it will not transmit the light. It should then be filtered, and a crystal of thymol added. It will not give very satisfactory staining reactions at first, and should be allowed to ripen at least a month or six weeks before using. It improves as a dye with every month that it is kept. Whenever hæmatoxyline has been made up with alum as in the above formula, an abundant reddish-brown precipitate forms after some time. This in no way interferes with the activity of the solution, but it must always be filtered before being used.

Barrett’s formula.—Introduced by Dr. W. H. Barrett, of Belfast. It gives almost as good results as the above. It is made from ordinary English extract of logwood, and is considerably cheaper.

The extract should be dried, and finely powdered, and then extracted with absolute alcohol for several days.

Filter and add slowly to

The strength of the solution will vary with different samples of logwood and must be estimated by trial. This solution is comparatively cheap and is useful for class purposes.

Ehrlich’s hæmatoxyline.—This very useful nuclear stain is made as follows:—

Add (a) slowly to (b) with constant agitation.

Allow to ripen in sunlight for two months before using. It may be employed as a rapid stain undiluted but far better results are obtained by using a weak solution, a few drops to a watch-glass full of distilled water, and staining slowly for from half an hour to two hours. The solution improves by keeping. If after a time the staining becomes diffuse it is an indication that the acetic acid has evaporated, and a few drops more should be added.